Project Summary Zinc finger nucleases ('ZFNs'), TAL effector nucleases '(TALENs'), CRISPR-Cas9 nucleases (?CRISPRs?) and meganuclease/TAL effector fusions ('MegaTALs', which are the focus of this project) are all highly specific nucleases that can generate single- or double-strand breaks at individual genomic loci. Each of these nuclease platforms is being developed for a wide variety of applications, including basic research, industrial and agricultural genome engineering, cellular therapeutics (for example, CAR T-cells), and direct gene therapy. Although CRISPR nucleases are now the system of choice for almost all genome engineering, their utility and performance for therapeutic applications is not a solved problem. For clinical use, nuclease performance is defined by the ease of its packaging and delivery, its activity and specificity in a living cell, and the balance of competing DNA repair outcomes. MegaTAL nucleases display several favorable properties for such purposes, including monomeric structures, small size, high activity and specificity, and unique cleavage mechanisms that produce 3' DNA overhangs. We have generated a large number of engineered MegaTAL nucleases and have described their ex vivo and in vivo performance in primary human cells and transgenic organisms, as summarized in the full text of this project description. While all these four of these platforms are being studied and used for gene therapy, optimization of their properties and behaviors (particularly to drive gene modification via homology-driven correction, rather than gene disruption via mutagenic end-joining) is an important ongoing priority. For any nuclease, the kinetics of DNA binding, cleavage and dissociation (and the corresponding affinity and half-life at each step) can alter the composition, structure and dynamic behavior of the DSB lesion in a manner that might affect each pathway differently. This can lead to significant differences in repair outcomes, as illustrated via our preliminary data. In this renewal application, we propose to leverage our engineered nuclease constructs and recently published results for two Specific Aims: (1) Determine the biophysical and enzymatic parameters of nuclease function that most strongly influence DNA repair outcomes and enhance gene modification via HDR. The overall premise for the first aim is that individual DNA repair pathways and their protein factors are uniquely sensitive to differences in the mechanisms and biophysical behaviors of the enzymes that generate a DSB. (2) Optimize our '2nd generation' of MegaTAL scaffolds (that are reduced in size and that appear to display improved activity and specificity) and corresponding mRNA delivery systems in genome editing directed towards primary hematopoietic stem cells (HSCs). The overall premise for the second aim is that the highly variable (but quite controllable) properties of MegaTALs and their delivery systems are particularly appropriate for assessing the efficiency of genome modification and subsequent persistence of gene edited primary cells, both in culture and upon transplantation and engraftment.